Abstract
We determined the structure of the Polα–primase complex trapped in a DNA elongation state. Cryo-electron microscopy showed that primase has a role in facilitating the timely termination of primer DNA elongation by Polα by hanging on the primer–template complex.
The question
Efficient high-fidelity genome replication is the foundation of healthy sustained life. Human primase and DNA polymerase alpha (Polα) form a multisubunit protein complex (Polα–primase, or primosome) to synthesize short RNA–DNA primers before handing them to Polε and Polδ for the start of leading-strand replication and the synthesis of millions of Okazaki fragments on the lagging strand1. We aimed to determine how the primosome engages chimeric RNA–DNA to coordinate transactions at the DNA replication fork. Structural determination of numerous intermediates is required to gain a deeper insight into these highly complicated dynamic processes. The structure of the Polα–primase complex trapped in a DNA elongation state is a crucial step toward this goal.
The discovery
The construction of short RNA–DNA primers by the primosome is a highly dynamic process that requires the protein complex to shapeshift several times. The intrinsic flexibility of the complex and the labile interactions used by the enzyme during primer synthesis make the structural determination of the primosome a challenge.
We used a two-pronged approach to solve these problems. First, we trapped the complex in its DNA elongation state by providing it with a dead-end primer–template substrate that we strategically designed. Second, we used cryo-electron microscopy (cryo-EM) single-particle analysis to deal with the heterogeneity and conformational flexibility of the protein complex. This combined strategy synchronized Polα–primase into a single conformational state with sufficiently low heterogeneity and flexibility such that cryo-EM single-particle analysis could determine the high-resolution structures of the human Polα–primase DNA elongation complexes.
We determined two cryo-EM DNA elongation-state structures from a single reconstituted biochemical sample. We named these structures elongation complex I and II (EC-I and EC-II, respectively) (Fig. 1a,b). EC-I is a subset of EC-II, a complex that has all four subunits of Polα–primase and the engaged primer–template substrate. Notably, in both complexes, the C-terminal domain of the large primase subunit (p58, also known as PRIM2) continues to hang on to the RNA primer and DNA template after handing over the substrate to its DNA polymerase counterpart (p180, also known as POLA1). This role of the C-terminal domain of p58 (p58C) in the DNA elongation process has not been reported previously.
Fig. 1 |. Cryo-EM structures of the human Polα–primase (primosome) DNA elongation complexes.
a,b, Cryo-EM structures of Polα–primase trapped in its two major DNA elongation states: EC-I (a) and the four-subunit EC-II (b). Both complexes revealed continued engagement of the primer–template substrate by the C-terminal domain of the p58 large primase subunit (p50C). EC-II revealed new interactions between p180 and the primase domain (p49–p58) that are important to ensure timely RNA–DNA primer termination.
The EC-II structure revealed an extensive rearrangement of the primosome conformation compared to its apo-state2. Interactions between p180 and the primase domain support this new conformation. Mutagenesis analysis showed that these elongation state-specific interactions are important for prompt primer termination, as is the p58C domain.
The implications
How the primosome consistently makes short primers for DNA replication remains a mystery. Our findings provide a structural explanation for how Polα–primase achieves prompt primer termination. Because the primosome has no proofreading function, overly long primers made by the enzyme would introduce unnecessary DNA mutations during genome replication. Furthermore, the many contacts between p180 and the rest of the enzyme would impose a physical constraint on the rotational demand of DNA double-helix formation during primer DNA elongation.
Although our structure-guided biochemical studies connect the newly identified interactions to p180 primer termination, their functional contributions and cellular function(s) remain to be tested. We still do not know the sequence of interactions the enzyme uses as it cycles through the entire primer synthesis process. For example, we trapped the enzyme in a single DNA elongation state, but our cryo-EM structural efforts resulted in two elongation complex structures. Whether the smaller complex is a remnant subcomplex from the larger one due to harsh sample preparation conditions or if it hints at a dynamic process governing primer termination is unclear.
Delineating the role of p58C in each function is a challenge and will be the next direction of the investigation, especially for understanding p180 primer termination. Additional structures captured at other biochemically related states would provide insights into how the enzyme changes shape during primer synthesis and how these changes connect to the preinitiation state conformation3.
EXPERT OPINION.
“The paper helps us understand the elongation step of the primer synthesis process. It shows the primosome as it simultaneously engages with the 3′ end of the primer via the p180 polymerase subunit and the 5′-triphosphate end of the RNA primer via its p58C subunit. Both interactions have already been described; the novelty of the current work is that it shows how the two crucial interactions can take place within the primosome.” Luca Pellegrini, University of Cambridge, Cambridge, UK.
BEHIND THE PAPER
In 2016, we prepared an RNA primer containing a 5′-triphosphate and demonstrated that it stays bound to p58C after the initiation of primer synthesis2,4. On the basis of these data, we proposed that the primer–template–p58C complex coordinates several transactions within Polα–primase and at the replication fork. To examine this, we developed a new method for 3′-DNA–RNA-5′-triphosphate primer synthesis5 and used it to prepare primosome complexes for X-ray crystallography structural determination. We got tiny needle-like crystals that were frustratingly time-consuming to improve. Then, in 2020, we began collaborating with Ci Ji Lim and his research group at the University of Wisconsin-Madison to use single-particle cryo-EM analysis to solve the structure of primosome DNA elongation complexes. The result of this fruitful collaboration is our reported cryo-EM structures of human primosome DNA elongation complexes. This work was supported by NIH NIGMS grants R00GM131023 to C.L. and R35GM127085 to T.H.T. T.H.T.
REFERENCES
- 1. Arezi B & Kuchta RD Eukaryotic DNA primase. Trends Biochem. Sci. 25, 572–576 (2000). A review article summarizing transactions within the eukaryotic Polα–primase complex.
- 2. Baranovskiy AG et al. Mechanism of concerted RNA–DNA primer synthesis by the human primosome. J. Biol. Chem. 291, 10006–10020 (2016). This paper reports crystal structures of human primosome in apo-form and p58C in complex with a primer–template, and reports biochemical studies of RNA primer length control and its switch from primase to Polα.
- 3. He Q et al. Structures of the human CST–Polα–primase complex bound to telomere templates. Nature 608, 826–832 (2022). This paper reports the cryo-EM structure of CST–Polα–primase bound to a telomere template in its preinitiation state, and reveals how CST organizes the DNA template and Polα–primase to stimulate RNA primer synthesis.
- 4. Baranovskiy AG et al. Insight into the human DNA primase interaction with template-primer. J. Biol. Chem. 291, 4793–4802 (2016). This paper reports how the primer–template interacts with different primase domains and which parts of the primer–template are essential for those interactions.
- 5. Baranovskiy AG et al. Insight into RNA–DNA primer length counting by human primosome. Nucleic Acids Res 50, 6264–6270 (2022). This paper reports the role of primosome linkers in the termination of primer synthesis and describes a method for preparing a chimeric RNA–DNA primer with a 5′-triphosphate.